|
 
- 积分
- 302
- 威望
- 302
- 包包
- 311
|
本帖最后由 qgjin 于 2010-5-7 04:30 编辑
1 D9 ^0 S& p5 K8 ]
, E6 N; U' D! z% q- z8 SPREPARATION OF MEF CULTURES2 N- C* \3 W) p* y$ X
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
" V5 M" `. F5 K5 G" wplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.: d% M9 B% z8 y- Z/ _, B% {
2. Mince the embryonic tissue in 5 ml of trypsin solution, transfer to an Erlenmeyer flask containing a stir bar and a few 5mm glass beads, and stir on
& q W A4 i; s( Pmagnetic stirrer for 15–25 min (use shorter incubation times if the embryos are E13 and use longer incubation times if the embryos are E16). Ideally,
2 L: T! K! E7 M! f+ r& sthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).0 N" L4 u1 B. `- o3 S4 g [( `. h
3. Pipette the cells with a 2-ml glass pipette to achieve cell suspension. Filter the suspension through a sieve or a screen, add 10 ml of
! n, s p. i8 f. b& w6 NMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.0 m4 r: X: p. p& Q
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%5 D2 a( n7 d9 n% _3 e+ s
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.$ N! Z( I! g9 n7 W
4. Plate the cell suspension onto cell culture plates at a density of about 2*106 cells per 100 mm plate (P0; i.e., passage 0) and incubate in
5 m5 |& ^! h9 D( P6 ~( i: VMEF growth medium at 5% CO2 and 37℃ for 24 h.
0 }4 M2 y O% P0 R5 B+ r5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
8 D) }% g) [6 M0 f7 d0 K6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
: [' V$ h- R8 Q1 I7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.+ d s# B6 }' z9 Y9 o2 e2 L
8. Add trypsin solution to the culture plates and incubate for 1–2 min.
" g1 V. u( q; R+ h' g @" D9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).( d6 q; j& S$ [; x! j, P
10. Freeze any MEFs not needed immediately.) [- ^! @, y# H) n% P, P. o
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. / Y* ?. z r; T; Z% }
11. Continue to passage cells as described in Steps 6–9 of this Box. MEFs at passages 2–4 are most suitable as feeder layer for cultivation of4 G' C r" _# G2 }! W1 }! U# O
undifferentiated maGSCs; prepare as follows.& _, _+ J6 z8 S7 u6 n3 d# w; A! r, p, K
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h. ?* p$ c; Z3 @4 [' P
13. Aspirate the mitomycin C solution and wash three times with PBS.
6 }- n$ T6 l7 C4 E! V8 U1 j14. Trypsinize MEFs as described in Steps 7–9 of this Box and replate them on new gelatin (0.1%)-treated microwell plates or to Petri dishes
" @+ ?$ Q. R8 R: @at a density of 50,000–60,000 cells/ cm2.
o8 d& J2 q) u t2 Z -CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to9 Y6 d8 \% s5 r; q$ f( U
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
6 T/ k3 c* K; d. B+ Cpassaging and thawing). |
-
总评分: 威望 + 5
包包 + 10
查看全部评分
|
发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
